Transmissible spongiform encephalopathies (TSEs) consist of a unique group of invariably fatal neurological disorders which affect both human and animals. TSEs are characterized by long presymptomatic incubation periods of months or years. Brain lesions associated with deposits of protease-resistant proteins are a hallmark of clinically manifest TSE disease.
Variant Creutzfeldt-Jakob disease (vCJD) is a rare and fatal human neurodegenerative condition. The classical form, that is to say CJD, presents as a subacute dementia, evolving over weeks to several months and is accompanied by pyramidal, extrapyramidal, and cerebellar signs. The mean age of death is 57 years but the disease may occur in the late teens and early twenties. In the final stages of the disease, there is an incapacitating dementia, usually with severe myoclonus. Recently, a variant of CJD (vCJD) was described in UK and France with distinct clinical and pathological features which affected younger individuals (average age 2-9 years, as opposed to 65 years).
As with CJD, vCJD is classified as a TSE because of characteristic spongy degeneration of the brain and its ability to be transmitted. The presumed infectious agent of TSE is often termed PrPsc or PrPres denoting the disease-specific form of the prion protein (PrP). The biological properties of PrPsc in vCJD and BSE share common biological properties e.g. they have similar incubation periods in various kinds of mice and hamsters. TSEs are also known in other animals. For instance, scrapie affects sheep and goats and has been found in many sheep-producing countries throughout the world for over 50 years. Chronic Wasting Disease (CWD) is a contagious fatal TSE in cervids (members of the deer and elk family).
The hypothesis of a link between vCJD and BSE was first raised because of the association of these two TSEs in time and place. More recent evidence supporting a link including identification of pathological features similar to vCJD in brains of macaque monkeys inoculated with BSE. A vCJD-BSE link is further supported by the demonstration that vCJD is associated with a molecular marker that distinguishes it from other forms of CJD and which resembles that seen in BSE transmitted to a number of other species. Studies of the distribution of the infectious agent in the brains of mice artificially infected with tissues from humans with vCJD and cows with BSE showed nearly identical patterns. The most recent and powerful evidence comes from studies showing that the transmission characteristics of BSE and vCJD in laboratory mice are almost identical, strongly indicating that they are due to the same causative agent. In conclusion, the most likely cause of vCJD is exposure to the BSE agent, most plausibly due to dietary contamination by affected bovine central nervous system tissue.
It follows that with regard to the human food chain there is an urgent need for methods to assess a possible TSE infection in the living host. Particularly there is a desire to assess TSE in living animals at a pre-symptomatic stage and before the animals are slaughtered and processed to enter the human food chain. One way to assess TSE is commonly applied when testing slaughtered cattle routinely for bovine spongiform encephalopathy. A brain sample is derived from a sacrificed animal and the presence of PrPsc/PrPres in the sample is assessed by means of an immunological test such as an ELISA or a Western blot. Apart from assays which target the TSE-associated form of the prion protein there are several approaches to assess TSE disease using surrogate markers. An example for surrogate markers are proteins other than PrP which are disclosed in WO 99/04237.
An alternative approach targets TSE disease specific changes of gene expression. Neurodegeneration in the brain is apparently closely linked with alterations among the RNA transcripts in nervous tissues. In addition, alterations in expression can be present in other body tissues as the proposed way of infection is thought to lead through the gut and the bloodstream to cross the blood brain barrier into the central nervous system. It has been found by the inventors that determination, that is to say qualitative or quantitative detection of a disease-specific target nucleic acid, requires a special kind of purification providing nucleic acids in high quality. Particularly, nucleic acid yield has to be high enough for detection of even very small amounts of target nucleic acid. In addition, degradation is to be minimized and high purity is desired.
Established products for nucleic acid purification which are commercially available include the MAGNA PURE instrument for which a variety of nucleic acid purification kits are on the market. In addition, stabilization reagents for biological samples are known to the art, e.g. the RNA/DNA stabilization reagent for blood/bone marrow distributed by Roche Diagnostics GmbH (Mannheim, Germany; Catalogue no. 11934317). With these means at hand, current methods appear to be suited for many routine purposes. However, the inventors noticed that the preparation of nucleic acids including high quality RNA from whole blood samples required optimization for particular detection purposes.
In view of nucleic acids as surrogate markers Begic, L. et al., Medicinski Arhiv (2002) 56(5-6):305-311 discussed differential analysis of transcripts with alternative splicing (DATAS), among other approaches, to identify RNAs as molecular markers to provide a tool for the diagnosis of TSE.
The DATAS method aims at identifying in a selected tissue or cell population one or more disease-specific RNAs in the form of a splicing variant. Thus, the presence or absence of the splicing variant in the tissue or cell population is correlated with the diagnosis of TSE. Ideally, the occurrence of the splicing variant is restricted to TSE infected individuals. However, other experimental settings based on quantitative determination of a splicing variant are also possible. In the latter case the presence of the splicing variant at either a higher or a lower level in the tissue or cell population is taken as an indicator of TSE infection.
Using the DATAS method as described in WO 99/46403 a number of marker nucleotide sequences were disclosed in WO 02/074986. Marker sequences were obtained using experimentally infected mice and scrapie infected sheep. Another marker nucleotide sequence identified likewise by means of the DATAS method is disclosed in WO 2004/050908. In both cases the marker sequence was validated using a panel of blood samples from BSE-infected cattle. Methods to probe for marker sequences included Northern hybridization, deoxyribonucleic acid (DNA) microarray hybridization, and quantitative PCR.
WO 2005/049863 discloses further nucleic acid sequences as diagnostic markers in the assessment of subacute spongiform encephalopathies from biological samples. As previously, the sequences have been identified in blood cells using the DATAS method. In order to evaluate the particular usefulness of these sequences the inventors set out to establish an assay aimed at the requirements of routine use and enhanced sample throughput. To this end, it was an object of the invention to provide an optimized workflow for sampling and sample preparation, specific oligonucleotide primer pairs for amplification of the marker nucleotide sequences by means of the polymerase chain reaction (PCR), and the detection of specific amplification products. It was another object of the invention to set up a PCR assay format which provides for a quick and reliable outcome of the diagnostic assay. Further, it was an object of the invention, to optimize test performance.